Source: Molica, F. et al., Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation. J. Vis. Exp. (2017)
This video demonstrates turbidimetric analysis to assess collagen-induced platelet aggregation in pre-treated, washed human platelets. Control wells show collagen-induced aggregation, whereas a high concentration of inhibitors blocks Pannexin-1 channels, thereby inhibiting collagen-induced platelet aggregation, evident from unchanged turbidity.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation
2. Blood Collection
3. Preparation of Human Washed Platelets
4. Aggregometry
Figure 1: Aggregometry. (A) Representative image of glass cuvettes (containing a stirring magnet) used for aggregometry. The cuvette on the left shows resting platelets while the cuvette on the right illustrates platelet aggregates after collagen-induced activation. (B) Representative image of an 8-well aggregometer for turbidimetric measurements. The wells used (asterisk) to incubate platelet suspensions present in a glass cuvette at 37 °C, and those used for the measurements (white arrow) are indicated in C.
The authors have nothing to disclose.
Citric acid monohydrate (C6H8O7*H2O) | Roth | 5949-29-1 | danger of eye damage/irritation |
Trisodium citrate dihydrate (Na3C6H5O7*2H2O) | Sigma-Aldrich | S1804 | – |
D(+)-glucose | Sigma-Aldrich | G8270 | – |
Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | – |
Potassium chloride (KCl) | Sigma-Aldrich | P9541 | – |
Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S6014 | – |
Sodium dihydrogenophosphate monohydrate (NaH2PO4*H2O) | Sigma-Aldrich | S9638 | – |
Magnesium chloride hexahydrate (MgCl2*6H2O) | Sigma-Aldrich | M9272 | – |
Calcium chloride hexahydrate (CaCl2*6H2O) | Sigma-Aldrich | 442909 | danger of eye damage/irritation |
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes) | ThermoFisher Scientific | 15630 | – |
Human serum albumin | CSL Behring | 00257/374 | – |
Hydrochloric acid | Sigma-Aldrich | 320331 | Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages. |
Eppendorf 5810 R | Fisher Scientific | – | – |
Heparin | Drosspharm AG/SA | 20810 | |
Prostacyclin I2 (PGI2) | Cayman | 18220 | – |
Apyrase from potatoes | Sigma-Aldrich | A6535 | – |
Fibrinogen (Haemocomplettan) | CSL Behring | HS 73466011 | – |
Thrombo-aggragometer SD-Medical | SD-Innovation | TA8V | – |
Brilliant blue FCF (Erioglaucine disodium salt) | Sigma-Aldrich | 80717 | Harmful to aquatic life with long lasting effects (Avoid release to the environnement) |
Collagen | Horm, Nycomed | – | |
Arachidonic acid | Bio/Data corporation | C/N 101297 | – |
Cell counter Sysmex KX-21N | Sysmex Digitana | – | – |
HEPES | Gibco | 15630-056 | – |
Glass cuvettes | SD-Innovation | THCV1000 | – |
Magnetic stirrers | SD-Innovation | THA100 | – |